Fig 2: miR-150 inhibits the cell cycle of nasopharyngeal carcinoma cells. (A) Flow cytometric analysis of the indicated nasopharyngeal carcinoma cells. Each bar represents the mean values ± SD of three independent experiments. *P<0.05. (B) Real-time PCR analysis of CCND1, CCND2, CKD2 and CCNE2 expression in the indicated cells. Transcript levels were normalized by GAPDH expression. Each bar represents the mean values ± SD of three independent experiments. *P<0.05. (C) Western blot analysis of CCND1, CCND2, CDK2, CCNE2 and p-Rb expression in the indicated cells. (D) Real-time PCR analysis of CDK4, CDK6, CCND3 and CCNE2 mRNA expression in overexpressed miR-150 and anti-miR-150 NPC cells, and their respective control vectors. U6 was used as the loading control. Each bar represents the mean values ± SD of three independent experiments. *P<0.05. (E and F) Relative Rb activity and E2F reporter activity in the indicated cells. Each bar represents the mean values ± SD of three independent experiments. *P<0.05.

Fig 3: EMP3 blocks S-phase entry and DNA replication.A EMP3, CCNE2, CDK2, P21, and PCNA expression in MDA-MB-231 cells was detected by western blot. B Cell cycle of MDA-MB-231 cells was analyzed by flow cytometry. Mean ± SD, n = 3. **P < 0.01, vs. shCtrl. C DNA replication of MDA-MB-231 cells was analyzed by EDU staining. Mean ± SD, n = 3. **P < 0.01, vs. shCtrl. D Viability of MDA-MB-231 cells was analyzed by CCK8. Mean ± SD, n = 3. ***P < 0.001, vs. shCtrl. E EMP3, CCNE2, CDK2, P21, and PCNA expression in MCF7 cells was detected by western blot. F Cell cycle of MCF7 cells was analyzed by flow cytometry. Mean ± SD, n = 3. **P < 0.01, vs. OE Ctrl. G Viability of MCF7 cells was analyzed by CCK8. Mean ± SD, n = 3. ***P < 0.001, vs. OE Ctrl.

Fig 4: CCND1, CCND2, CDK2 and CCNE2 mediates the miR-150-downregulation-induced NPC cell growth. (A) Individual silencing of CCND1, CCND2, CDK2 and CCNE2 rescued the proliferation rate enhanced by anti-miR-150. (B) Individual silencing of CCND1, CCND2, CDK2 and CCNE2 rescued the colony-formation ability enhanced by anti-miR-150. (C) Individual silencing of CCND1, CCND2, CDK2 and CCNE2 rescued the anchorage-independent growth ability enhanced by anti-miR-150.

Fig 5: Clinical relevance of miR-150 with CCND1, CCND2, CDK2 and CCNE2 in nasopharyngeal carcinoma tissues. (A) miR-150 expression in eight freshly-frozen human nasopharyngeal carcinoma tissues. Transcript levels were normalized by U6 expression. One tissue sample was randomly selected and numbered as T1, other 7 tissue samples were numbered in order as T2, T3, T4, T5, T6, T7 and T8. The expression levels of miR-150 in each NPC tissue were normalized by the corresponding miR-150 expression in T1 NPC tissue. The relative expression levels of miR-150 after normalization were used to perform the correlation analysis among miR-150 and CCND1, CCND2, CKD2 and CCNE2 expression. (B) Analysis of CCND1, CCND2, CDK2 and CCNE2 expression in eight freshly-frozen human nasopharyngeal carcinoma tissues. Transcript levels were normalized by GAPDH expression. One tissue sample was randomly selected and numbered as T1, other 7 tissue samples were numbered in order as T2, T3, T4, T5, T6, T7 and T8. The expression levels of CCND1, CCND2, CKD2 and CCNE2 in each NPC tissue were normalized by the corresponding CCND1, CCND2, CKD2 and CCNE2 expression levels in T1 NPC tissue. The relative expression levels of CCND1, CCND2, CKD2 and CCNE2 after normalization were used to perform the correlation analysis among miR-150 and CCND1, CCND2, CKD2 and CCNE2 expression. (C) Correlation between miR-150 levels and CCND1, CCND2, CDK2 and CCNE2 expression in nasopharyngeal carcinoma tissues.